Chronic inflammation-mediated bone loss occurs due to increased osteoclastic bone resorption, mediated largely by the increased local production of pro-inflammatory cytokines. TNFalpha is a major inflammation mediator and is both directly and indirectly involved in the production of osteoclasts. At present, although the anti-TNF drugs represent a major therapeutic advance, the efficacy of these agents is somewhat clouded by the fact that many RA patients who receive significant relief from these drugs continue to suffer progressive joint erosion. In this proposal, we plan to further investigate TNFalpha effects on osteoclast-mediated bone loss and will test the following hypotheses: 1) TNFalpha promotes osteoclastogenesis by priming the differentiation of progenitors into the osteoclast lineage, thus increasing the precursor pool; 2) RANK signaling is essential for TNFalpha-induced osteoclastogenesis in vivo; and 3) TNFalpha sustains mature osteoclast survival through a Src-dependent and RANK-independent mechanism, thus promoting enhanced resorption. To examine hypothesis 1, we will use hTNF-Tg transgenic mice to determine the effects of TNFalpha on osteoclast precursor numbers, the duration of TNFalpha-mediated priming and the effect of anti-TNF therapy on this priming. To test hypothesis 2, we will use three in vivo models: a) we will generate and analyze hTNF-Tg/RANK-/- mice to determine if chronic TNFalpha over-expression can compensate for the absence of RANK signaling; b) transfer bone marrow cells from RANK-/- mice to lethally irradiated hTNF-Tg mice; and c) treat hTNF-Tg mice with RANK:Fc and examine them for the presence of osteoclasts. To examine hypothesis 3, we will block the activity of Src family kinases by various approaches and use osteoclasts from src-/- mice to evaluate the effects of TNFalpha and TNF blockade on their survival. hTNF-Tg/src-/- mice will be generated and treated with RANK:Fc to determine the effect on osteoclast depletion. These studies will provide definitive in vivo evidence as to the mechanism of TNFalpha-induced osteoclastic bone resorption and also provide pre-clinical data that may help develop a therapeutic intervention for erosive diseases.